1. Field of the Invention
The present invention relates generally to the fields of neurobiology and biochemistry. More particularly, it concerns methods and compositions for therapeutic intervention against Parkinson""s disease. In particular, methods of making and sequestering dopamine are disclosed.
2. Description of Related Art
Parkinson""s disease (PD) is an age-related disorder characterized by a loss of dopamine neurons in the substantia nigra of the midbrain. These neurons have the basal ganglia as their major target organ. The symptoms of PD include tremor, rigidity and ataxia. The disease is progressive, but can be treated by replacement of dopamine through the administration of pharmacological doses of the precursor for dopamine, L-3,4-dihydroxyphenylalanine (levodopa; L-DOPA; Marsden, 1986; Vinken et al., 1986). However, with chronic use of this pharmacotherapy, the patients often develop a fluctuating response to L-DOPA. There are many suggested mechanisms for the development of the fluctuation, but one simple explanation is that the patients reach a threshold of cell and terminal loss, wherein the remaining cells cannot synthesize and store sufficient dopamine from the precursor.
Typically PD patients are routinely treated with a combination of L-DOPA and a DOPA decarboxylase inhibitor such as carbidopa or benserazide. Unfortunately, after an initial period of satisfactory, smooth and stable clinical benefit from L-DOPA therapy, lasting on the average 2-5 years, the condition of many patients deteriorates and they develop complex dose-related, as well as unpredictable, response fluctuations. The causes of the response fluctuations are probably multiple and complex, but pharmacokinetic problems likely play some role. There is a correlation between the clinical fluctuations and the oscillations of L-DOPA plasma levels. However, many of the problems are a result of the unfavorable pharmacodynamics response to L-DOPA, i.e., a short half-life in vivo due to various central factors.
Another treatment route is through intracerebral grafting. PD is the first disease of the brain for which therapeutic intracerebral grafting has been used in humans. Preclinical and clinical data indicate that transplanted cells (the graft) used in cell transplantation protocols for these types of neurodegenerative diseases survive and integrate with the host tissue, and provides functional recovery (Wictorin et al., 1990).
Several attempts have been made to provide the neurotransmitter dopamine to cells of the basal ganglia of Parkinson""s patients by transplantation of fetal brain cells from the substantia nigra, an area of the brain rich in dopamine-containing cell bodies and also the area of the brain most affected in PD. Fetal dopaminergic neurons have been shown to be effective in reversing the behavioral deficits in rat models of PD induced by selective dopaminergic neurotoxins (Bjorklund et al., 1986; Dunnett et al., 1983). The major effect of fetal transplantation in PD has been in enhancing patients"" response to L-DOPA, rather than alleviating the need for the drug. This effect is thought to be due to added capacity to decarboxylate L-DOPA and storage of the formed dopamine (Lindvall et al, 1994; Sawle et al, 1992).
Non-fetal cell transplants also have been used in an attempt to combat PD, e.g., the use of chromaffin cells. A major advantage of this type of transplantation protocol is that the graft source is not a fetal source and thus, circumvents the ethical and logistical problems associated with acquiring fetal tissue. Using the chromaffin cell protocol, normalization of behavior has been observed. However, the functional recovery of this behavior is temporary and the animals revert to their pre-transplantation status (Bjorklund and Stenevi, 1985; Lindvall et al., 1987). The inability of this type of treatment protocol to maintain normal behavioral activity in animals in the PD model renders clinical application of this protocol as well as other treatment therapies ineffective.
Finally, it is known that the chemical deficit that results in PD is the inability to supply dopamine. The rate limiting enzyme in the production of dopamine, tyrosine hydroxylase, has been cloned and the anatomical localization of the affected region has been identified as the basal ganglia. Neverthless, in animal models, it has been shown that increasing the activity of tyrosine hydroxylase in Parkinsonian tissue does not result in the long-term amelioration of the symptoms of PD.
Clearly, there is a need for methods and compositions that will correct the chemical deficit in PD in a reliable manner that will lead to the treatment of the disease.
The present invention provides a method for producing dopamine in a cell comprising transforming a cell with a first polynucleotide encoding aromatic L-amino acid decarboxylase (AADC) and a second polynucleotide encoding vesicular monoamine transporter (VMAT) under conditions suitable for the expression of AADC and VMAT, wherein the polynucleotides each are under the transcriptional control of a promoter active in eukaryotic cells; and contacting the cell with L-3,4-dihydroxyphenylalanine (L-DOPA), whereby AADC converts L-DOPA to dopamine and VMAT sequesters the dopamine in endosomes in the cell. In certain preferred embodiments, the first and second polynucleotides are covalently attached. It is contemplated that the first and second polynucleotides are part of a viral vector. In alternative embodiments, first and second polynucleotides are not covalently attached. It may be that the first and second polynucleotides are part of first and second viral vectors, respectively. It is contemplated that the viral vector may be selected from the group consisting of retrovirus, adenovirus, herpes virus, adeno-associated virus and lentivirus.
In those embodiments in which the first and second polynucleotides are covalently attached it is contemplated that the first and second polynucleotides may be under control of different promoters or under the control of the same promoter. In those embodiments in which the first and second polynucleotides are under the control of the same promoter, the first and second polynucleotides may be separated by an internal ribosome entry site.
It is contemplated that as used herein the promoter may be a tissue specific promoter, an inducible promoter or a constitutive promoter. Such promoters are described throughout the application and are well known to those of skill in the art. In particularly preferred embodiments, the promoter may be selected from the group consisting of CMV IE, SV40 IE, xcex2-actin, EF1-xcex1, a TH promoter, AADC non-neuronal promoter; an AADC promoter, a VMAT2 promoter region, a GTP cyclohydrolase I promoter and a dopamine transporter promoter. Of course these and the other promoters listed throughout the specification are exemplary promoters and one of skill in the art may well substitute other promoter regions for those listed herein and still achieve the objectives of the present invention.
In those embodiments in which the first and second polynucleotides are covalently linked, it is contemplated that the first and second polynucleotides each are covalently linked to a polyadenylation signal.
In particularly preferred embodiments, the cell is a fibroblast cell. In other embodiments the cell may be a fetal brain cell, astrocytes, neuronal stem cells, neuronal precursor cells, myoblasts, bone marrow stromal cells.
In certain aspects of the present invention, it is contemplated that the method further may comprise transforming the cell with a polynucleotide encoding tyrosine hydroxylase (TH) wherein the TH encoding polynucleotide is under the transcriptional control of a promoter. In still further embodiments, the method further may comprise transforming the cell with a polynucleotide encoding GTP cyclohydrolase I (GTPCH) in addition to the polynucleotide encoding TH, wherein the GTPCH polynucleotide is are under the transcriptional control of a promoter.
In specific embodiments, the cell may be transformed in vivo. In other embodiments, the cell is transformed ex vivo and implanted into a subject. In those embodiments in which the cell is implanted into a subject, the L-DOPA may be administered to the patient using any route commonly recommended by a physician. More particularly, it is contemplated that the L-DOPA may be administered orally, subcutaneously or sublingually. In certain defined embodiments, the cell is derived from the subject prior to transformation.
Another aspect of the present invention contemplates a method of treating Parkinson""s disease in a subject comprising obtaining a cells from the subject; transforming the cells with a first polynucleotide encoding L-amino acid decarboxylase (AADC) and a second polynucleotide encoding VMAT under conditions suitable for the expression of AADC and VMAT, wherein the polynucleotides each are under the transcriptional control of a promoter; implanting transformed cells into the subject; and providing L-DOPA to the subject, whereby AADC converts L-DOPA in vivo to dopamine and VMAT sequesters the dopamine in endosomes in the cell, which sequestered dopamine over a longer duration of time than from cells without storage of L-DOPA. In particularly preferred embodiments, the L-DOPA is administered orally, sublingually, subcutaneously, intravenously or by duodenal infusion.
It is contemplated that the dose of L-DOPA to be administered may fall within the range of any L-DOPA therapy currently in use or any concentration that becomes useful in future. It is particularly contemplated that the L-DOPA is administered in a dose of between about 50 mg to about 2500 mg of L-DOPA per day. Thus it is contemplated that 50 mg, 75 mg, 100 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1200 mg, 1250 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, 2500 mg of L-DOPA per day or more. This dose of L-DOPA may be administered in one dose or alternatively may be split into several (2, 3, 4, 5 or more) aliquots to be administered over the period of 24 hours. Alternatively, it also is contemplated that the L-DOPA may be administered continuously throughout the day. It is contemplated that the L-DOPA is administered alone or alternatively may be administered in combination with carbidopa and/or any other PD therapeutic agent as described in the specification below. In particularly preferred embodiments, the L-DOPA is administered in combination with carbidopa at a dose of between about 20 mg to about 300 mg carbidopa per day. Thus it is contemplated that 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg carbidopa per day or more may be administered. This may be administered in a single dose or in multiple doses throughout the day.
In specific embodiments, it is contemplated that the transformed cells are implanted via stereostaxic surgery.
Also contemplated by the present invention is a kit comprising a polynucleotide encoding aromatic L-amino acid decarboxylase (AADC); and a polynucleotide encoding vesicular monoamine transporter (VMAT), the polynucleotides being located in suitable container means therefor.
In preferred embodiments, the VMAT is VMAT type II. In other preferred embodiments, the VMAT is VMAT type I. In specifically defined embodiments, the polynucleotides each are part of a viral vector and each are under the transcriptional control of a promoter active in eukaryotic cells. In particular embodiments, the viral vector is selected from the group consisting of retrovirus, adenovirus, herpes virus, adeno-associated virus and lentivirus. In certain preferred embodiments the kit comprises L-DOPA in a suitable container means therefor.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.